Characterization of a conjugative IncA plasmid co-harbouring blaNDM-5 and blaVIM-1 in a KPC-producing Klebsiella pneumoniae clinical isolate K27

Abstract

Multiple-carbapenemase-producing Klebsiella pneumoniae has emerged as a critical threat to global health due to limited treatment options and high mortality. This study aimed to characterize the genomic features of a triple-carbapenemase-producing K. pneumoniae clinical isolate. A carbapenem-resistant K. pneumoniae (CRKP) strain K27 was collected from a patient in a tertiary hospital in January 2025. Antimicrobial susceptibility testing, carbapenemase detection, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), conjugation experiments, and plasmid fitness cost assays were performed. Whole-genome sequencing (WGS) of strain K27 was carried out on Illumina and PacBio platforms, bioinformatic analyses were performed using tools such as PlasmidFinder, ResFinder and ISfinder to characterize plasmid architecture, including replicon typing, resistance gene profiling, and mobile genetic element identification. Strain K27 was an extensively drug-resistant ST11 K. pneumoniae producing three carbapenemases (NDM, KPC and VIM). Both S1-PFGE and WGS revealed that strain K27 carried four plasmids. The bla KPC-2 gene was located on an IncFII pHN7A8 :IncR hybrid plasmid (assigned as pK27-KPC2) and carried by a truncated transposon Tn 6296 , while the bla NDM-5 and bla VIM-1 genes were co-harboured on a single IncA plasmid (assigned as pK27-NDM5/VIM1), where they were carried by a truncated transposon Tn 125 and integron In916, respectively. The two corresponding genetic elements were separated by IS CR1 , and this arrangement was highly similar to that of plasmid p24A20009_B_NDM. Conjugation experiments demonstrated that the plasmid pK27-NDM5/VIM1 was transferable at a frequency of (4.5 ± 1.2) × 10 −2 , and the resulting transconjugant was designated as K27-C. Independent sample t -tests further revealed significant differences between the transconjugant K27-C and the Escherichia coli J53Azi R groups, both in the area under the curve ( t = 3.63, p = 0.002) and in the maximum optical density ( t = 2.57, p = 0.02). The finding of two different class B metallo-carbapenemase genes co-harboured on a single conjugative IncA plasmid in a KPC-producing ST11 K. pneumoniae clinical isolate is alarming. This co-production of three distinct carbapenemases in a single strain could pose considerable challenges for the selection of clinical anti-infective therapies. Furthermore, the isolate may pose a high risk of subsequent clonal dissemination and/or horizontal gene transfer, necessitating ongoing epidemiological surveillance.